MicroRNA-34a-5p: A pivotal therapeutic target in gallbladder cancer

Gallbladder cancer incidence has been increasing globally, and it remains challenging to expect long prognosis with the current systemic chemotherapy. We identified a novel nucleic acid-mediated therapeutic target against gallbladder cancer by using innovative organoid-based gallbladder cancer models generated from KrasLSL-G12D/+; Trp53f/f mice. Using comprehensive microRNA expression analyses and a bioinformatics approach, we identified significant microRNA-34a-5p downregulation in both murine gallbladder cancer organoids and resected human gallbladder cancer specimens. In three different human gallbladder cancer cell lines, forced microRNA-34a-5p expression inhibited cell proliferation and induced cell-cycle arrest at the G1 phase by suppressing direct target (CDK6) expression. Furthermore, comprehensive RNA sequencing revealed the significant enrichment of gene sets related to the cell-cycle regulators after microRNA-34a-5p expression in gallbladder cancer cells. In a murine xenograft model, locally injected microRNA-34a-5p mimics significantly inhibited gallbladder cancer progression and downregulated CDK6 expression. These results provide a rationale for promising therapeutics against gallbladder cancer by microRNA-34a-5p injection, as well as a strategy to explore therapeutic targets against cancers using organoid-based models, especially for those lacking useful genetically engineered murine models, such as gallbladder cancer.


INTRODUCTION
The incidence of gallbladder cancer (GBC), a type of biliary tract cancer (BTC), has increased worldwide.However, because it is difficult to diagnose at an early stage due to the lack of clinical manifestations, 1 radical surgery is rarely performed.Moreover, although systemic chemotherapy has progressed, it is not sufficient to improve prognosis, and the 5-year survival rate remains <20%. 2 Recently, wholeexome sequencing has revealed a landscape of genomic alterations in BTC, and various altered genes, such as TP53, KRAS, SMAD4, ARID1A, and PIK3CA, have been identified in GBC. 3,4In some cases with NTRK gene fusion-positive tumors, mismatch repair-deficient tumors, or microsatellite instability-high tumors, NTRK inhibitors or immune checkpoint inhibitors are expected to exhibit a high treat-ment effect 5,6 ; however, patients with such gene alterations account for <10% of patients with BTC.Therefore, there is an urgent need to develop novel treatments, including molecular-targeted therapies, for patients with GBC.
][9][10] However, only a few targets applicable to clinical use have been identified.This limited success can be attributed to numerous factors such as the analysis of bulk samples, a high degree of intra-and intertumor heterogeneity, and the use of unsuitable control samples.In addition, the lack of an ideal genetically engineered murine model that accurately reflects human primary GBC has hampered research.In recent years, three-dimensional (3D) cultured organoid-based research has become widespread in cancer biology. 11his advanced in vitro culture tool allows for long-term culture of normal stem cells under physiological conditions 12 and provides a high-throughput drug screening platform with improved reliability compared to conventional 2D culture systems. 135][16] These organoid-based carcinogenesis models present considerable potential as innovative tools for exploring GBC biology.
With recent advances in nucleic acid-mediated therapies, novel therapeutic strategies targeting microRNAs (miRNAs) have been developed for various types of cancers, 17 including GBC. [18][19][20] However, the crucial miRNAs associated with GBC progression that could serve as high-priority therapeutic targets have not yet been identified.
In this study, we comprehensively examined the miRNA expression profiles of Kras-activated and Trp53-deleted tumorigenic GB organoids mimicking GBC and normal GB organoids mimicking the normal GB.We compared these profiles and identified pivotal therapeutic miRNA targets that regulate GBC progression.A direct gene target and comprehensive transcriptional changes after forced miRNA expression were determined, and the effects of local administration of the miRNA into mouse xenograft models were evaluated to develop a new therapeutic option against GBC.

miR-34a-5p is downregulated in GBC
To identify miRNAs that were differentially expressed between normal GB and GBC, we conducted a comprehensive miRNA expression analysis by comparing organoids mimicking normal GB and GBC.Although both types of organoids exhibited cyst formation (Figure 1A), Cre-mediated induction of Kras G12D and deletion of Trp53 were observed only in GBC organoids (Figures 1B and S6).Using qualified RNAs extracted from these organoids (Figure S1), the study revealed that the expression levels of 144 kinds of miRNAs and 91 miRNAs were higher (log 2 FC [fold change] > 1) and lower (log 2 FC < À1), respectively, in GBC organoids than in normal GB organoids in the miRNA microarray analyses (Figures 1C and 1D).When focusing on miRNAs with a signal intensity >25, the expression levels of 18 miRNAs were higher (log 2 FC > 1) and those of 19 miR-NAs were lower (log 2 FC < À1) in GBC organoids than in normal GB organoids.Although miR-21a-5p, a well-known oncogenic miRNA, was upregulated, four types of miRNAs-miR-34a-5p, miR-181a-5p, miR-378a-3p, and miR-205-5p, which have been implicated as tumor suppressors in various cancers [21][22][23][24][25] -exhibited decreased expression levels in GBC organoids (Figure 1E).Through validation of the expression levels of these four miRNAs by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), the downregulation of miR-34a-5p expression was confirmed, with the largest differences between normal GB and GBC organoids (Figure 1F).Similarly, in a public miRNA microarray database using human resected specimens (GSE104165), 26 miR-34a-5p expression levels showed the most significant decrease in GBC tissues compared to normal GB tissues (Figure 1G).Based on these findings, we hypothesized that miR-34a-5p plays a pivotal role in GBC and could serve as a promising candidate for miRNA-based targeted therapy for GBC.

Forced miR-34a-5p expression inhibits cell proliferation and viability in GBC cell lines
Three types of human GBC cell lines (G415, NOZ, and TGBC2TKB) were used for subsequent in vitro studies.To explore the potential of miR-34a-5p as a therapeutic target for GBC, these cell lines were transfected with an miR-34a-5p mimic.Significant inhibition of cell proliferation in cells transfected with the mimic was observed compared to that in the negative control in 2D cell cultures (NOZ, p < 0.01; G415 and TGBC2TKB, p < 0.05; Figure 2A) in a dose-dependent manner (Figure S2).These results suggest that forced miR-34a-5p expression can decrease the proliferation rate and viability of GBC cells.
Forced miR-34a-5p expression induces cell-cycle arrest at the G1 phase in GBC cell lines Mechanisms underlying these phenomena were determined by assessing the cell cycle.The miR-34a-5p mimic expression induced a remarkable increase in the percentage of cells in the G1 phase in all cell lines (Figure 2B).According to the miRDB database, CDK6, an important kinase for the G1/S transition, and CCND1, which forms a complex with and functions as a regulatory subunit of CDK6, 27 were identified as direct targets of miR-34a-5p (target scores: 92 and 58, respectively) (Figure 2C).The protein expression levels of CDK6 and cyclin D1 were substantially decreased in all three cell lines by forced expression of the miR-34a-5p mimic (Figures 2D and S6).These findings suggest that overexpression of miR-34a-5p induces cell-cycle arrest at the G1 phase, at least partly by downregulating the expression of CDK6 and cyclin D1, leading to cell proliferation inhibition in GBC cell lines.

CDK6 is a direct target of miR-34a-5p
To further explore whether the 3 0 UTR of CDK6 is a direct target of miR-34a-5p, luciferase-based reporters were used (Figure 2E).Cotransfection of NOZ cells with the reporter construct containing the wildtype (WT) or mutant-type (MT) CDK6 3 0 UTR sequences at the downstream of the luciferase gene and the miR-34a-5p mimic resulted in a significant decrease in luciferase activity compared to cells transfected with the negative control mimic, only when using the reporter construct with WT CDK6 3 0 UTR sequences (p < 0.01; Figure 2E).These results, together with the marked decrease in CDK6 expression in GBC cells transfected with the miR-34a-5p mimic (Figure 2D), indicated that miR-34a-5p predominantly inhibits the cell cycle and cell proliferation, partially via direct CDK6 downregulation in GBC cells.

Differentially expressed gene (DEG) enrichment related to cell cycle by miR-34a-5p expression in GBC cells
To determine the transcriptomic differences between control and miR-34a-5p mimic-transfected GBC cell lines, RNA samples from paired NOZ cells were used for RNA sequencing (RNA-seq) analyses.The principal-component analysis plot showed distinct clustering between NOZ cells transfected with miR-34a-5p and negative controls (Figure S3).Compared to the control, miR-34a-5p-transfected NOZ cells exhibited 2,437 significant DEGs, with 927 upregulated and 1,037 downregulated genes (Figure 3A).As expected, the downregulated genes showed the most significant association with the miR-34a-5p target genes, as determined by enrichment analysis and gene set enrichment analysis (GSEA) (Figure 3B, false discovery rate [FDR] = 9.7eÀ20; Figure 3C, p = 6.99eÀ08).Known miR-34a-5p target gene expressions such as Snail1 (target score: 94), BIRC5 (score not shown), 28 Notch2 (target score: 84) and CDK6 were significantly decreased (Figure S4).Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of these genes revealed a significant enrichment of biological processes related to the inhibition of the cell cycle and G1-to-S-phase transition (Figures 3D and 3E, adjusted p = 3.28eÀ08; Figure S5), which is consistent with the aforementioned results showing the inhibition of cell-cycle progression and proliferation in miR-34a-5p-transfected GBC cell lines.To confirm the suppressive function of miR-34a-5p in GBC in vivo, we used a GBC xenograft model.When the average size of the subcutaneously implanted tumors reached $80 mm 3 , a mixture of miR-34a-5p or a negative control with atelocollagen was injected around the tumors every 4-5 days for 3 weeks (Figure 4A).Compared to the negative controls, miR-34a-5p significantly inhibited GBC tumor growth after five injections (Figures 4A and 4B).miR-34a-5p injection significantly decreased the CDK6 expression levels in the tumors compared to those in the negative control (Figure 4C), suggesting that forced expression of miR-34a-5p by direct injection also has the potential to suppress GBC growth in vivo.

DISCUSSION
Because only a small number of therapeutic options are currently available for GBC, novel therapeutic development is urgently required.In this study, we uncovered the vital role of miR-34a-5p in GBC and determined that miR-34a-5p forced expression leads to efficient GBC progression suppression via cell-cycle inhibition, at least partly by CDK6 downregulation, a direct target of this miRNA.
The 3D organoid culture system has been established as a physiological model that closely mimics the structure and differentiation that occur in the body compared to 2D culture systems.In particular, these organoid models have enabled us to culture primary normal epithelial cells for a long time, including normal GB cells, and have therefore led us to precisely compare GBC and normal GB in vitro.Owing to the lack of genetically modified murine models for GBC presently, normal GB organoids and GBC organoids induced by Kras activation and Trp53 loss with Cre expression used here present attractive alternatives for GBC studies.Although the induction of an oncogene and/or the regulation of a tumor suppressor gene in normal epithelial cells does not necessarily lead to tumorigenicity, 16 it has already been confirmed that Cre-induced GBC organoids were actually tumorigenic when inoculated subcutaneously into mice, but normal GB organoids were not tumorigenic. 16Therefore, these organoids present promising tools for assessing the biology of GBC.
In this study, several miRNAs were found to be differentially expressed in GBC, including miR-34a-5p.Although miR-34a-5p is transcriptionally activated by TP53, 29,30 the miR-34a expression is also regulated by other mechanisms, such as CpG methylation of its promoter region, 31 sponge effects of long noncoding RNA, 32,33 and chromosomal deletion. 24Therefore, its expression is likely not solely dependent on p53 status but also on other factors.TP53 muta-tions are identified in over 40%-60% of GBC cases, establishing it as one of the predominant driver mutations for GBC. 3,34In addition, significantly decreased expression levels of miR-34a-5p were observed in most GBC tissues, as deduced from the results deposited in public databases, and the overexpression of miR-34a-5p resulted in growth inhibition in GBC cell lines, even those with WT TP53, such as G415 cells.Further examination using GB and GBC organoids with WT p53 may be necessary to determine the biological significance of miR-34a-5p in GBC in such cases.miR-34a-5p is downregulated in a wide range of solid tumors and hematological malignancies. 25Specifically, miR-34a-5p directly regulates several target mRNAs encoding proteins related to cell-cycle transition (CCND1, CDK6, Notch1, and Notch2), 27,35 apoptosis (Bcl-2 and BIRC5), [36][37][38][39] migration, and invasion (Snail and Notch1), [40][41][42][43] resulting in the repression of tumor progression.In the context of GBC, only a few studies have reported the relationship between miR-34a-5p and GBC.For example, the low expression of miR-34a, which extends telomere length, is a useful biomarker for predicting poor prognosis in patients with GBC. 19In this study, we revealed that miR-34a-5p overexpression, as a therapeutic option, induced strong gene set enrichment of cell-cycle regulators with decreased expression levels related to cell proliferation, the epithelial-mesenchymal transition, and survival (e.g., CDK6, BIRC5, Snail1, and Notch2) in GBC cell lines and murine xenograft models.Thus, we strongly suggest that GBC cell growth inhibition by miR-34a-5p can aid in the development of therapeutic strategies for treating aggressive GBC.
Regarding the clinical application of miR-34a-5p supplementation, a liposomal miR-34a mimic, MRX34, has been developed, and the results of a Phase I study, in which patients with advanced solid tumors received MRX34 intravenously, were reported in 2020. 44lthough the disease control rate was 29% in that study, the trial was closed early owing to four patient deaths with unexpected severe immune-mediated toxicities.Thus, specific drug delivery systems (DDSs) that avoid or minimize nonspecific delivery to other normal tissues require further elucidation. 45Alternatively, in current clinical practice, endoscopic ultrasonography-guided injections can be performed for GB lesions. 46Therefore, local miR-34a-5p supplementation, with or without systemic chemotherapy, may be a realistic option for clinicians to apply the results obtained in clinical settings.
In conclusion, although further optimization is required, our in vitro and in vivo analyses revealed that forced expression of miR-34a-5p presents a promising therapeutic option for patients with GBC.

Organoid-based GB carcinogenesis model
Cre-transduced and pLKO.1-, a negative control construct, transduced GB organoids derived from Kras LSL-G12D/+ ; Trp53 f/f mice (a gift from Dr. Yoshitaka Hippo, Chiba Cancer Center Research Institute, Chiba, Japan).Detailed protocols for establishment and characterization have been described previously. 16Briefly, the GB was isolated from Kras LSL-G12D/+ ; Trp53 f/f mice, and normal GB organoids were established from these cells.Lentivirus-expressing Cre cells were transduced into GB organoids in vitro to establish GBC organoids.The tumorigenicity of the Cre-transduced GB organoids was confirmed by subcutaneously inoculating the organoids into nude mice, after which aggressively growing nodules were observed.Furthermore, the nodules were excised, and subsequent organoid cultures were established after the dissociation of the recovered tumor-derived epithelial cells.These tumor-derived organoids were used as Cre-transduced GB organoids to model GBC. 16dvanced DMEM/F12 (Thermo Fisher Scientific, Waltham, MA) media containing L-glutamine, penicillin, streptomycin, and amphotericin B, supplemented with 50 ng/mL epidermal growth factor (Peprotech, Rocky Hill, NJ), 100 ng/mL Noggin (Peprotech), 1 mM Jagged1 (AnaSpec, Fremont, CA), and 10 mM Y27632 (Wako, Osaka, Japan), were used with the Matrigel (no.354234, Corning, Corning, NY) to culture organoids.

Comprehensive miRNA expression analyses
For analysis, the Matrigel was lysed with Cell Recovery Solution (BD Biosciences, San Jose, CA) and washed with PBS to collect pure, viable organoid populations.Total RNA was extracted from these organoids using the 3D-Gene RNA Extraction Reagent (Toray Industries, Tokyo, Japan), and quality checked using an Agilent RNA 6000 Pico Kit and an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA).Comprehensive miRNA expression analyses were performed using the 3D-Gene miRNA Labeling Kit and the 3D-Gene Mouse miRNA Oligo Chip (version 21; Toray Industries), as previously described. 47Fluorescent signals were scanned using a 3D-Gene Scanner 3000 and analyzed using 3D-Gene Extraction software (Toray Industries).The global normalization method was applied to background-subtracted signal intensities, setting the median of these signal intensities to 25.0.The FC values of Cre-transduced GB organoids for each miRNA were calculated using signals from pLKO.1-transducedGB organoids as reference.

Cell culture and miRNA transfection
Human GBC cell lines G415, NOZ, and TGBC2TKB were obtained from Tohoku University (Sendai, Japan), the Japanese Collection of Research Bioresources cell bank (Osaka, Japan), and the RIKEN cell bank (Tsukuba, Japan), respectively.The NOZ cell line was established by Dr. S. Nagamori (National Institute of Infectious Diseases, Tokyo, Japan). 48G415 cells were cultured in RPMI 1640 (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS), NOZ cells in Williams' Medium E (Thermo Fisher Scientific) supplemented with 10% FBS and L-glutamine, and TGBC2TKB cells in DMEM (Thermo Fisher Scientific) containing low glucose and supplemented with 5% FBS.All of the cells were cultured in a humidified atmosphere containing 5% CO 2 at 37 C.

2D cell culture viability and cell proliferation assays
Cell proliferation and viability were determined in 2D cells using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.Briefly, treated cells were seeded in 96-well plates at a density of 2 Â 10 3 or 2.5 Â 10 3 cells/well, depending on the cell line.After 24, 48, 72, and 96 h of incubation, MTT (0.5 mg/mL in the medium) was added to each well.The cells were then incubated for 3 h at 37 C, and the purple-blue formazan precipitate was dissolved using 200 mL DMSO.Absorbance was measured at 570 nm using a microplate reader (MULTISKAN GO, Thermo Fisher Scientific).Media containing the MTT reagent but without cells were used as a blank control.All of the experiments were performed in triplicate (minimum).

Cell-cycle analysis
The treated cells were collected and fixed in 70% ethanol at 4 C for 2 h.The cells were then washed with PBS and stained with 20 mg/mL propidium iodide containing 0.25 mg/mL RNase for 30 min in a dark environment at 37 C. Finally, the cells in each cell-cycle phase were assayed using a MACS Quant Analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany), and the percentage of cells in the G1, S, and G2/M phases was determined using FlowJo version 10 software (BD Biosciences, Ashland, OR).

qRT-PCR
Total RNA was extracted from the organoids or cell lines using an miRNeasy Micro Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions.RNA samples were reverse transcribed using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) with TaqMan MicroRNA Assay (Thermo Fisher Scientific).Subsequently, qPCR was performed using the TaqMan Fast Advanced Master Mix (Applied Biosystems) and the LightCycler 96 Real-Time PCR System (Roche, Basel, Switzerland) in 96-well plates.The analysis of relative gene expression data was calculated using the 2 ÀDDCq method. 49All of the reactions were performed in duplicate.

Immunohistochemistry (IHC)
Organoid Matrigels were first depolymerized by Cell Recovery Solution and then embedded in iPGell (GenoStaff, Tokyo, Japan), followed by formalin fixation.Harvested subcutaneous tumor specimens were formalin fixed, paraffin embedded, and sectioned at 4 mm.H&E staining was used for histological analysis.For IHC analysis, tissue sections were deparaffinized and soaked in 0.3% H 2 O 2 in methanol at room temperature for 10 min to block endogenous peroxidase activity.Antigen retrieval was performed by heating the specimens in 10 mM sodium citrate buffer (pH 6.0) using a microwave.After three 5-min washes with PBS, the tissue sections were incubated with a primary antibody against CDK6 (sc-7961; Santa Cruz Biotechnology, Dallas, TX) at room temperature for 30 min (1:200 dilution).After another three 5-min washes with PBS, the sections were incubated with secondary anti-mouse IgG (K4001; Agilent Technologies, Santa Clara, CA) for 30 min at room temperature.3,3 0 -Diaminobenzidine+ (K3468, Agilent Technologies) was used as the chromogen, and the nuclei were counterstained with Mayer's hematoxylin.CDK6 IHC slides were scanned using an Axio Scan.Z1 (Zeiss, Jena, Germany).The resulting whole-slide images were imported into an open-source software program (QuPath version 0.3.2) for viewing and assessment.Furthermore, a digital assistance tool was developed using QuPath's positive cell detection algorithm and used for the assessment. 50

RNA-seq
Total RNA was isolated from NOZ cells treated with 5 nM miR-34a-5p mimic or negative control mimic 48 h after transfection.Three biological replicates were used for each sample (n = 3).The RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies), and RNAs with an RNA integrity number above nine were processed for sequencing.Libraries were constructed using the NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs, Ipswich, MA) according to the manufacturer's instructions.Then, 150-bp paired-end sequencing was performed using an Illumina NovaSeq 6000 instrument (Illumina, San Diego, CA).Raw sequence data were filtered using Fastp (version 0.19.5) to remove adapter sequences and low-quality or short reads.The filtered data were aligned with the human reference genome (GRCh38.p13)using the STAR software (version 2.7.10a).Gene expression counts were summed using RSEM software (version 1.3.1).
The DEGs obtained from RNA-seq-based expression profiling were analyzed using the integrated Differential Expression and Pathway analysis online tools.DESeq2 results were used for differential expression analysis, and genes with an FDR < 0.01 and an absolute FC > 2 identified by DESeq2 were designated DEGs.The DEGs were then used to generate a heatmap after converting the data to base two logarithms and Z scores.The heatmap function of the ComplexHeatmap package (version 2.14) was used with R software (version 4.2.1;https:// www.r-project.org/).The relationship between DEGs and miRNAs was assessed using miRTarBase, 51 and enrichment analysis was conducted using ShinyGO 0.77. 52GSEA was performed using the cluster-Profiler R package, KEGG pathway, and WikiPathway databases. 53al luciferase reporter assay Plasmids were constructed using the pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega, Madison, WI) for the binding site in the 3 0 UTR of the potential target gene (CDK6) based on the miRNA target prediction database, miRDB.54 For the reported gene assay, NOZ cells (5 Â 10 3 cells) were cotransfected with reporter vectors (CDK6 WT or CDK6 MT), miR-34a-5p mimic, and negative control mimic using Lipofectamine 3000 Reagent (Invitrogen) on the Corning 96 Half Area Well Solid White Flat Bottom Polystyrene Tissue Culture-treated microplates (no.3688; Corning).Following transfection for 72 h, luciferase activity was evaluated using the Nano-Glo Dual-Luciferase Reporter Gene Assay System (Promega) and GloMax Discover Microplate Reader (Promega).Normalization was performed using Renilla luciferase as the reference standard.All of the experiments were conducted in triplicate.

In vivo experiments in mice bearing human tumor xenograft
For the in vivo model, NOZ cells (3 Â 10 6 cells) were subcutaneously injected into the flank regions of 6-week-old BALB/c nu/nu mice.Ten days later, local treatment with synthetic miRNAs prepared using atelocollagen as the DDS was initiated.Briefly, a mixture of miRNA-atelocollagen was prepared using either 1 nmol of the miR-34a-5p (HMI0508; Sigma-Aldrich, Saint Louis, MO) or 1 nmol of the negative control miRNA (HMC0003; Sigma-Aldrich) and an AteloGene Local Use "Quick Gelatin" kit (KOKEN, Tokyo, Japan), according to the manufacturer's protocol. 55,56The mixture was injected around the tumor site five times over 3 weeks.The tumor volume was calculated using the following formula: V = A Â B 2 /2 (mm 3 ), where A is the largest diameter (mm) and B is the smallest diameter (mm).At 35 days after tumor cell implantation, the mice were euthanized, and the tumors were collected for further analysis.

Bioinformatics analyses
To investigate the miRNA expression in human tissue, we searched the GEO database for datasets using the keywords "gallbladder carcinoma" and "miRNA."We used the GSE104165 dataset and the GEO2R online analysis tool to determine the expression levels of miRNAs of interest in GBC and normal tissues.The targets of these miRNAs were identified using the miRDB online database. 54

Statistical analyses
All of the statistical analyses were performed using JMP Pro 15.1.0(SAS Institute, Cary, NC) or GraphPad Prism 9.3.1 (GraphPad, San Diego, CA).Group comparisons were performed using the Kruskal-Wallis, Mann-Whitney U, Pearson chi-square, or Wilcoxon rank-sum tests.All of the tests were two-sided.p < 0.05 were considered statistically significant.

Figure 2 .
Figure 2. miR-34a-5p inhibits the cell proliferation of GBC cells in 2D cell cultures (A) Relative proliferation rate in human GBC cell lines after transfection with miR-34a-5p mimics was significantly suppressed compared to that in the negative control using an MTT assay.Data are presented as the means ± SDs (n = 3).*p < 0.05; **p < 0.01.(B) Cell-cycle arrest at the G1 phase was induced in GBC cell lines after transfection with miR-34a-5p mimics by flow cytometry.Representative images from 3 independent experiments are shown.(C) Potential target sequences in the 3 0 UTR of cyclin D1 and (legend continued on next page)

Figure 3 .
Figure 3. Enrichment of gene sets related to cell-cycle regulation in the miR-34a-5p mimic-transfected GBC cells (A) Heatmap showing hierarchical clustering based on DEG expression in miR-34a-5p-transfected cells (n = 3) and controls (n = 3).DEGs were identified with an FDR < 0.01 and an absolute FC >2.(B) The downregulated genes exhibited significant enrichment in the miR-34a-5p target gene set, as evidenced by enrichment analyses (FDR = 9.7eÀ20).The most enriched miRNA target genes along with their FDR and gene counts are listed.(C) GSEA plot demonstrating the enrichment of miR-34a-5p target gene sets in miR-34a-5p-transfected GBC cells compared to the negative control (p = 6.99eÀ08).(D) GSEA results demonstrate significant enrichment of gene sets related to the cell cycle following the miR-34a-5p-transfection.The most enriched biological processes, along with their p values and gene counts, are presented.(E) GSEA plot demonstrating the enrichment of cell-cycle-related gene sets in miR-34a-5p-transfected GBC cells compared to the negative control (adjusted p = 3.28eÀ08).

Figure 4 .
Figure 4. Local miR-34a-5p administration suppresses GBC growth in vivo (A) Subcutaneous miR-34a-5p mimic or negative control with atelocollagen injection around the NOZ tumor was repeated every 4 or 5 days, for a total of 5 sessions.Error bars indicate the mean ± SD; n = 5 mice per group.(B) miR-34a-5p mimics significantly suppressed tumor growth compared to the negative control.Macroscopic images of tumors from each group after 5 injections over 21 days are shown.The scale bar indicates 10 mm.(C) IHC staining of CDK6 protein expression (brown in the nucleus) in harvested subcutaneous tumors after 5 injections for 21 days (left).The scale bar indicates 50 mm.The injection of miR-34a-5p mimics significantly decreased the expression levels of CDK6 protein.Error bars represent the mean ± SD (n = 3 in each group) (right).*p < 0.05.